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And Lytic Granule Exocytosis Microtubule-Organizing Center CD8+ Tumor-Infiltrating T Cells Are Deficient in Perforin-Mediated Cytolytic Activity Due to Defective Microtubule-Organizing Center Mobilization This information is current as and Lytic Granule Exocytosis of September 24, 2021. Sasa Radoja, Masanao Saio, David Schaer, Mythili Koneru, Stanislav Vukmanovic and Alan B. Frey J Immunol 2001; 167:5042-5051; ; doi: 10.4049/jimmunol.167.9.5042 Downloaded from http://www.jimmunol.org/content/167/9/5042 References This article cites 39 articles, 16 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/167/9/5042.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 24, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. CD8؉ Tumor-Infiltrating T Cells Are Deficient in Perforin-Mediated Cytolytic Activity Due to Defective Microtubule-Organizing Center Mobilization and Lytic Granule Exocytosis Sasa Radoja,* Masanao Saio,* David Schaer,† Mythili Koneru,* Stanislav Vukmanovic,† and Alan B. Frey1* Tumor-infiltrating lymphocytes (TIL) are well known to be functionally impaired typified by the inability to lyse cognate tumor cells in vitro. We have investigated the basis for defective TIL lytic function in transplantable murine tumor models. CD8؉ TIL are nonlytic immediately on isolation even though they express surface activation markers, contain effector phase cytokine mR- Downloaded from NAs, and contain perforin and granzyme B proteins which are packaged into lytic granules. Ag-specific lytic capability is rapidly recovered if purified TIL are briefly cultured in vitro and tumor lysis is perforin-, but not Fas ligand mediated. In response to TCR ligation of nonlytic TIL in vitro, proximal and distal signaling events are normal; calcium flux is rapid; mitogen-activated protein/extracellular signal-related kinase kinase, extracellular regulatory kinase 2, phosphoinositide-3 kinase, and protein kinase C are activated; and IL-2 and IFN-␥ is secreted. However, on conjugate formation between nonlytic TIL and cognate tumor cells in vitro, the microtubule-organizing center (MTOC) does not localize to the immunological synapse, thereby precluding exocytosis http://www.jimmunol.org/ of preformed lytic granules and accounting for defective TIL lytic function. Recovery of TCR-mediated, activation-dependent MTOC mobilization and lytic activity requires proteasome function, implying the existence of an inhibitor of MTOC mobilization. Our findings show that the regulated release of TIL cytolytic granules is defective despite functional TCR-mediated signal transduction. The Journal of Immunology, 2001, 167: 5042–5051. rowth of cancer in the authochthonous host elicits anti- Human antitumor immune responses do not usually correlate tumor immune response and is detected by both T and B with tumor remission but has motivated considerable research in cell antitumor immune responses (1). For example, im- cancer immunotherapy. Although a variety of protocols have G by guest on September 24, 2021 mune cells infiltrating human tumors are seen in many different proved successful in immunization of rodents to resist primary tumor types and, although the prognostic significance of infiltrates tumor challenges, only rarely have large preexisting tumors been 2 varies among tumor type, tumor-infiltrating lymphocytes (TIL) eradicated (6). The failure to cure established rodent tumors and contain tumor-specific T cells. Patients often also have tumor-re- the modest success of human experimental immunotherapies, to- active T cells in lymph nodes draining tumor sites and in the cir- gether with the realization that antigenic human cancers elicit im- culation. These sources of T cells have been used to study the Ag mune response but are usually not eliminated, has sparked interest specificity and frequency of T cell antitumor immune response and in understanding the basis for inadequate antitumor T cell immune to prepare T cell lines used both in experimental adoptive immu- responses (7). notherapy (2) and as the basis for tumor Ag identification by ex- The presence of TIL in human cancers demonstrates that tumor pression gene cloning (3). Humoral antitumor immune response Ags are expressed in situ that are capable of eliciting antitumor T has also been noted in many human cancers (4), and patient sera cell immune response, which nonetheless fails to eliminate tumors have similarly been useful in identification of tumor Ags, most ϩ (8). The lytic function of CD8 TIL has been investigated in var- recently by the SEREX method (serological analysis of recombi- ious human (9) and murine (10, 11) tumors and found to be de- nant tumor cDNA expression libraries; Ref. 5). Patient humoral fective. Deficient lytic function is transient because, on purification and T cell responses prove that human cancers are able to prime from tumor cells and culture in vitro, tumor-specific killing can be tumor-specific immune responses. Similar studies using experi- detected (12). mental models have proven the immunogenicity of rodent tumors. We have investigated the basis for defective TIL lytic function in transplantable murine tumor models. Freshly isolated TIL, al- Departments of *Cell Biology and †Pathology, New York University School of Medi- though nonlytic in vitro, contain mRNAs encoding effector phase cine, New York, NY 10016 cytokines, serine esterases, and perforin characteristic of cytolytic Received for publication June 22, 2001. Accepted for publication August 22, 2001. effector T cells. Furthermore, perforin and granzyme B proteins are The costs of publication of this article were defrayed in part by the payment of page synthesized and packaged into lytic granules showing that TIL are charges. This article must therefore be hereby marked advertisement in accordance ϩ with 18 U.S.C. Section 1734 solely to indicate this fact. mature CD8 T cells. TIL recover perforin-mediated, Ag-specific 1 Address correspondence and reprint requests to Dr. Alan B. Frey, Department of lysis if purified and cultured briefly in vitro. After recovery of lytic Cell Biology, New York University School of Medicine, Room MSB 690, 550 First function, on conjugate formation with cognate tumor cells in vitro, Avenue, New York, NY 10016. E-mail address: [email protected] TIL mobilize cytotoxic granules to the immunological synapse, 2 Abbreviations used in this paper: TIL, tumor-infiltrating lymphocyte; AICD, acti- vation-induced cell death; MTOC, microtubule-organizing center; PKC, protein ki- whereas granules in freshly isolated, nonlytic TIL do not migrate. nase C; FasL, Fas ligand; PI3 kinase, phosphatidylinositol 3-kinase. In addition, the microtubule-organizing center (MTOC) does not Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 5043 localize to the immunological synapse in nonlytic TIL. This defect Conjugate formation and intracellular staining for fluorescence precludes movement of lytic granules to the immunological syn- microscopy apse and accounts for defective TIL lysis. Biochemical analyses CD8ϩ TIL (6 ϫ 105) were mixed with tumor cells (3 ϫ 105) in 0.6 ml demonstrated that TCR kinase-mediated signaling is functional in serum-free RPMI 1640 at room temperature. Cells were centrifuged at freshly isolated, nonlytic TIL. Our findings show that after arrival 170 ϫ g for 10 min to promote conjugate formation, incubated for 5–10 to the tumor microenvironment, antitumor T cells are unable to min at 37°C, and transferred to coverslips (VWR Scientific, South Plain- release cytolytic granules on contact with tumor which may permit field, NJ) pretreated with 1% Alcian blue. Cells were fixed in 4% parafor- maldehyde for 30 min at room temperature, permeabilized with 0.01% tumor escape from T cell lysis. saponin, 0.5% BSA in PBS (30 min at room temperature), and then then stained with 0.5 ␮g/ml of mouse monoclonal anti-␤-tubulin Ab (Boehr- inger Mannheim, Indianapolis, IN), mouse anti-Rab 27a (BD Transduction Materials and Methods Laboratories, San Diego, CA), rabbit anti-PYK-2 (a gift from J. Schless- Mice inger, New York University School of Medicine), or goat polyclonal anti- granzyme B Ab (Santa Cruz Biochemicals, Santa Cruz, CA) (1 h at 37°). Male C57BL/6 mice were obtained from The Jackson Laboratory (Bar After three washes (0.01% saponin, 0.5% BSA in PBS), cells were incu- Harbor, ME). Mice were housed four per cage in a barrier facility and bated with secondary Ab (FITC-conjugated anti-mouse IgG for ␤-tubulin maintained on a 12-h light/dark cycle (7 a.m. to 7 p.m.) with ad libitum and Rab 27a or PE- or FITC-conjugated anti-goat for granzyme B) (30 min access to food and water. A sentinel program revealed that the mice were at RT°), washed three times (0.01% saponin, 0.5% BSA in PBS), incubated
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